Publications

• Enzymes of hydrogen metabolism in Pyrococcus furiosus.

  Silva P.J., van den Ban E.C., Wassink H., Haaker H., de Castro B., Robb F.T., Hagen W.R.

  The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni-Fe hydrogenase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two ... >> More

  The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni-Fe hydrogenase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni-Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe-4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe-4S] protein. With P. furiosus soluble [4Fe-4S] ferredoxin as the electron donor the membranes produce H2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H2 evolution to H2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with Em approximately -0.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni-Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H2) connected by devices for transduction, transfer, recovery and safety-valving of energy. << Less

  Eur. J. Biochem. 267:6541-6551(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8-A resolution.

  Peters J.W., Lanzilotta W.N., Lemon B.J., Seefeldt L.C.

  A three-dimensional structure for the monomeric iron-containing hydrogenase (CpI) from Clostridium pasteurianum was determined to 1.8 angstrom resolution by x-ray crystallography using multiwavelength anomalous dispersion (MAD) phasing. CpI, an enzyme that catalyzes the two-electron reduction of t ... >> More

  A three-dimensional structure for the monomeric iron-containing hydrogenase (CpI) from Clostridium pasteurianum was determined to 1.8 angstrom resolution by x-ray crystallography using multiwavelength anomalous dispersion (MAD) phasing. CpI, an enzyme that catalyzes the two-electron reduction of two protons to yield dihydrogen, was found to contain 20 gram atoms of iron per mole of protein, arranged into five distinct [Fe-S] clusters. The probable active-site cluster, previously termed the H-cluster, was found to be an unexpected arrangement of six iron atoms existing as a [4Fe-4S] cubane subcluster covalently bridged by a cysteinate thiol to a [2Fe] subcluster. The iron atoms of the [2Fe] subcluster both exist with an octahedral coordination geometry and are bridged to each other by three non-protein atoms, assigned as two sulfide atoms and one carbonyl or cyanide molecule. This structure provides insights into the mechanism of biological hydrogen activation and has broader implications for [Fe-S] cluster structure and function in biological systems. << Less

  Science 282:1853-1858(1998) [PubMed] [EuropePMC]

• A simple energy-conserving system: proton reduction coupled to proton translocation.

  Sapra R., Bagramyan K., Adams M.W.

  Oxidative phosphorylation involves the coupling of ATP synthesis to the proton-motive force that is generated typically by a series of membrane-bound electron transfer complexes, which ultimately reduce an exogenous terminal electron acceptor. This is not the case with Pyrococcus furiosus, an arch ... >> More

  Oxidative phosphorylation involves the coupling of ATP synthesis to the proton-motive force that is generated typically by a series of membrane-bound electron transfer complexes, which ultimately reduce an exogenous terminal electron acceptor. This is not the case with Pyrococcus furiosus, an archaeon that grows optimally near 100 degrees C. It has an anaerobic respiratory system that consists of a single enzyme, a membrane-bound hydrogenase. Moreover, it does not require an added electron acceptor as the enzyme reduces protons, the simplest of acceptors, to hydrogen gas by using electrons from the cytoplasmic redox protein ferredoxin. It is demonstrated that the production of hydrogen gas by membrane vesicles of P. furiosus is directly coupled to the synthesis of ATP by means of a proton-motive force that has both electrochemical and pH components. Such a respiratory system enables rationalization in this organism of an unusual glycolytic pathway that was previously thought not to conserve energy. It is now clear that the use of ferredoxin in place of the expected NAD as the electron acceptor for glyceraldehyde 3-phosphate oxidation enables energy to be conserved by hydrogen production. In addition, this simple respiratory mechanism readily explains why the growth yields of P. furiosus are much higher than could be accounted for if ATP synthesis occurred only by substrate-level phosphorylation. The ability of microorganisms such as P. furiosus to couple hydrogen production to energy conservation has important ramifications not only in the evolution of respiratory systems but also in the origin of life itself. << Less

  Proc. Natl. Acad. Sci. U.S.A. 100:7545-7550(2003) [PubMed] [EuropePMC]

• Purification and characterization of a membrane-bound hydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

  Sapra R., Verhagen M.F., Adams M.W.

  Highly washed membrane preparations from cells of the hyperthermophilic archaeon Pyrococcus furiosus contain high hydrogenase activity (9.4 micromol of H(2) evolved/mg at 80 degrees C) using reduced methyl viologen as the electron donor. The enzyme was solubilized with n-dodecyl-beta-D-maltoside a ... >> More

  Highly washed membrane preparations from cells of the hyperthermophilic archaeon Pyrococcus furiosus contain high hydrogenase activity (9.4 micromol of H(2) evolved/mg at 80 degrees C) using reduced methyl viologen as the electron donor. The enzyme was solubilized with n-dodecyl-beta-D-maltoside and purified by multistep chromatography in the presence of Triton X-100. The purified preparation contained two major proteins (alpha and beta) in an approximate 1:1 ratio with a minimum molecular mass near 65 kDa and contained approximately 1 Ni and 4 Fe atoms/mol. The reduced enzyme gave rise to an electron paramagnetic resonance signal typical of the so-called Ni-C center of mesophilic NiFe-hydrogenases. Neither highly washed membranes nor the purified enzyme used NAD(P)(H) or P. furiosus ferredoxin as an electron carrier, nor did either catalyze the reduction of elemental sulfur with H(2) as the electron donor. Using N-terminal amino acid sequence information, the genes proposed to encode the alpha and beta subunits were located in the genome database within a putative 14-gene operon (termed mbh). The deduced sequences of the two subunits (Mbh 11 and 12) were distinctly different from those of the four subunits that comprise each of the two cytoplasmic NiFe-hydrogenases of P. furiosus and show that the alpha subunit contains the NiFe-catalytic site. Six of the open reading frames (ORFs) in the operon, including those encoding the alpha and beta subunits, show high sequence similarity (>30% identity) with proteins associated with the membrane-bound NiFe-hydrogenase complexes from Methanosarcina barkeri, Escherichia coli, and Rhodospirillum rubrum. The remaining eight ORFs encode small (<19-kDa) hypothetical proteins. These data suggest that P. furiosus, which was thought to be solely a fermentative organism, may contain a previously unrecognized respiratory system in which H(2) metabolism is coupled to energy conservation. << Less

  J. Bacteriol. 182:3423-3428(2000) [PubMed] [EuropePMC]

• The nitrogen-fixing complex of bacteria.

  Zumft W.G., Mortenson L.E.

  Biochim Biophys Acta 416:1-52(1975) [PubMed] [EuropePMC]

• The structure and mechanism of iron-hydrogenases.

  Adams M.W.

  Hydrogenases devoid of nickel and containing only Fe-S clusters have been found so far only in some strictly anaerobic bacteria. Four Fe-hydrogenases have been characterized: from Megasphaera elsdenii, Desulfovibrio vulgaris (strain Hildenborough), and two from Clostridium pasteurianum. All contai ... >> More

  Hydrogenases devoid of nickel and containing only Fe-S clusters have been found so far only in some strictly anaerobic bacteria. Four Fe-hydrogenases have been characterized: from Megasphaera elsdenii, Desulfovibrio vulgaris (strain Hildenborough), and two from Clostridium pasteurianum. All contain two or more [4Fe-4S]1+,2+ or F clusters and a unique type of Fe-S center termed the H cluster. The H cluster appears to be remarkably similar in all the hydrogenases, and is proposed as the site of H2 oxidation and H2 production. The F clusters serve to transfer electrons between the H cluster and the external electron carrier. In all of the hydrogenases the H cluster is comprised of at least three Fe atoms, and possibly six. In the oxidized state it contains two types of magnetically distinct Fe atoms, has an S = 1/2 spin state, and exhibits a novel rhombic EPR signal. The reduced cluster is diamagnetic (S = 0). The oxidized H cluster appears to undergo a conformation change upon reduction with H2 with an increase in Fe-Fe distances of about 0.5 A. Studies using resonance Raman, magnetic circular dichroism and electron spin echo spectroscopies suggest that the H cluster has significant non-sulfur coordination. The H cluster has two binding sites for CO, at least one of which can also bind O2. Binding to one site changes the EPR properties of the cluster and gives a photosensitive adduct, but does not affect catalytic activity. Binding to the other site, which only becomes exposed during the catalytic cycle, leads to loss of catalytic activity. Mechanisms of H2 activation and electron transfer are proposed to explain the effects of CO binding and the ability of one of the hydrogenases to preferentially catalyze H2 oxidation. << Less

  Biochim Biophys Acta 1020:115-145(1990) [PubMed] [EuropePMC]